title: High-speed positional cloning based on restriction landmark genome scanning.
abstract: Restriction landmark genome scanning (RLGS) was developed as a method of genome analysis that is based on the concept that restriction enzyme sites can be used as landmarks. In this article, we demonstrate how this method can be used for the systematic, successful positional cloning of mouse mutant reeler gene. The major advantage of the RLGS method is that it allows the scanning of several thousand spots/loci throughout the genome with one RLGS profile. High-speed positional cloning based on the RLGS method includes (1) high-speed construction of a linkage map (RLGS spot mapping), (2) high-speed detection of RLGS spot markers tightly linked to the mutant phenotype (RLGS spot bombing method), and (3) construction of YAC contigs covering the region where tightly linked spot markers are located (RLGS-based YAC contig mapper). We introduced a series of these procedures by using them to positionally clone the reeler gene. High-speed construction of the whole genetic map and spots/loci (less than 1 cM) within the closest flanking markers is demonstrated. The RLGS-based YAC contig mapper also efficiently yielded the YAC physical contig map of the target region. Finally, we cloned the reeler gene, which is the causal gene for the perturbation of the three-dimensional brain architecture due to the abnormal migration of neuroblasts in reeler mouse. Since the RLGS method itself can be used for any organism, we conclude that the total RLGS-based positional cloning system can be used to identify any mutant gene of any organism.
authors.affiliation: Genome Science Laboratory, Institute of Physical and Chemical Research, (RIKEN), Tsukuba, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Okazaki
authors.forename: Y
authors.initials: Y
authors.lastname: Hayashizaki
mesh_terms: D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000302:isolation & purification*; D005819:Genetic Markers; D016678:Genome; D051379:Mice; D009154:Mutation; D015183:Restriction Mapping / Q000379:methods*
chemical_list: D005819:Genetic Markers; D004247:DNA